X-ray diffraction analysis of human adenoviruses

性状（由申请方提供）：腺病毒（Ad）是呼吸道、眼部和胃肠道疾病的主要病原体。复制缺陷型和条件复制型Ad载体也被用于大量的人类基因转移试验以及抗微生物疫苗的开发。我们提出的研究利用了最近在确定3.5 ½分辨率下人类腺病毒的第一个晶体结构方面取得的进展和获得的技术/专业知识。尽管获得了重要的结构见解，但在主要衣壳蛋白（六邻体）以及辅助蛋白的精细X射线图和高分辨率cryoEM结构之间发现了许多显著差异。此外，我们仍然缺乏详细的知识的关键水泥蛋白，PVI，牵连在细胞进入过程中的膜渗透的位置。我们建议解决水泥蛋白的结构和身份方面的差异，阐明关键的蛋白质-蛋白质的相互作用，稳定的Ad衣壳获得一个精致的模型HAdV，使用重原子标记的Met/Cys残基和/或通过对接到病毒的电子密度图的模块化结构域的辅助蛋白，独立地确定在高分辨率。此外，我们将通过进行诱变和感染性研究来评估改变的Ad衣壳的结构-功能关系。1）准确鉴定稳定天然AdV衣壳的辅助蛋白和相关蛋白-蛋白相互作用，并评估影响顶点区域稳定性的条件; 2）分析水泥蛋白缺失突变体的结构-功能关系以及修饰的Ad衣壳在基于载体的基因递送中的有效性;确定代表腺病毒装配早期阶段的未成熟腺病毒颗粒的结构。这些调查和随后的比较分析，它们的结构应该提供更好的理解的相互作用，稳定的腺病毒颗粒和结构-功能关系的改变的广告类型，具有独特的生物物理和生化特性。

DESCRIPTION (provided by applicant): Adenoviruses (Ad) are major causative agents of respiratory, ocular and gastrointestinal diseases. Replication-defective and conditionally replicating Ad vectors are also being employed in a significant number of human gene transfer trials as well as for the development of anti-microbial vaccines. Our proposed studies capitalize on the recent progress made and technology/expertise obtained in determining the first crystal structure of human adenovirus at 3.5 ¿ resolution. Even though significant structural insights were gained, a number of notable differences were found between the refined x-ray maps and the high-resolution cryoEM structures of the major capsid protein (hexon) as well as accessory proteins. Moreover, we still lack detailed knowledge of the location of the key cement protein, PVI, implicated in membrane penetration during cell entry. We propose to resolve the discrepancies with regards to the structure and identities of cement proteins, elucidate the critical protein-protein interactions that stabilize the Ad capsid by obtaining a refined model of HAdV, using heavy atom labeling of Met/Cys residues and/or by docking into the virus electron density maps the modular domains of accessory proteins, determined independently at high resolution. In addition, we will evaluate structure-function relationships of the altered Ad capsid by performing mutagenesis and infectivity studies. Specifically, this proposal will accomplish the above goals by: 1) accurately identifying the accessory proteins and the associated protein-protein interactions that stabilize the native AdV capsid and evaluate the conditions that affect the stability of the vertex region; 2) analyzing the structure-function relationships of the deletin mutants of cement proteins and the effectiveness of the modified Ad capsids in vector-based gene delivery and 3) determining the structure of immature AdV particles that represent an early stage of adenovirus assembly. These investigations and subsequent comparative analysis of their structures should provide greater understanding of the interactions that stabilize the adenovirus particle and structure-function relationships of the altered Ad types, which possess unique biophysical and biochemical characteristics.